Protein synthesis is an integral part of gene expression, and translational control mechanisms are known to operate during the initiation phase of the process. The projects proposed here are designed to elucidate the molecular events of initiation, with special emphasis on the synthesis and mechanism of action of the three initiation factors IF1, IF2, and IF3. First, the expression of initiation factor genes in Escherichia coli will be studied by recombinant DNA techniques. The genes for IF1 (infA), IF2 (infB) and IF3 (infC) already have been cloned, mapped and sequenced. We propose to test whether or not IF1 is required for cell viability, by inactivating infA on the bacterial chromosome. Operon and protein fusion of infA and lac will be used to determine whether or not infA expression is regulated by IF1 levels (autogenous regulation) or by the stringent response. The coordinate regulation of all three initiation factor genes as a function of growth rate also will be studied in vivo will lac fusions. Overproducing strains for initiation factors will be constructed and isolation methods will be developed that employ fast protein liquid chromatography to purify the proteins. Second, we propose to study in vivo the physiological consequences of reducing the cellular levels of each of the initiation factors. In initial studies with IF2, infB was placed under control of the lac promoter/operator, and expression was regulated by the concentration of the inducer, IPTG. Strains with infA and infC under lac promoter control will be constructed and we shall study the effects of reduced factor levels on protein synthesis rates, polysome profiles, rRNA operon expression, specific proteins synthesized, and initiation pathway intermediates. Third, the mechanism of action of initiation factors will be probed by in vitro mutagenesis and by fluorescence polarization techniques. Both random and site-directed mutagenesis will be employed and the resulting mutated genes will be screened in vivo by transformation of the genes into strains carrying the chromosomal gene under lac promoter control. Growth characteristics in the absence of IPTG will depend on the mutated factor. Interesting mutant forms will be overexpressed and the mutated factor purified and studied by a variety of in vitro assays for function. Continuation of fluorescence polarization studies of factor- ribosome interactions will address quantitatively the binding of individual initiation factors to 30S and 70S initiation complexes in order to shed light on the reaction pathway of initiation. The studies should result in an increase in our knowledge of the initiation process, which is essential for understanding mechanisms of protein synthesis and translational control at the molecular level.